ONCOLOGY REPORTS 32: 2635-2647, 2014

Anticancer effects of β-elemene in gastric cancer cells and its potential underlying : A proteomic study

JUN-SONG LIU, SHI-CAI HE, ZHENG-LIANG ZHANG, RUI CHEN, LIN FAN, GUANG‑LIN QIU, SHUAI CHANG, LIANG LI and XIANG-MING CHE

Department of General Surgery, The First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China

Received July 4, 2014; Accepted August 26, 2014

DOI: 10.3892/or.2014.3490

Abstract. Gastric cancer is a common malignancy with a Introduction poor prognosis. β-elemene is a broad-spectrum anticancer drug extracted from the traditional Chinese medicinal herb Gastric cancer is the fourth most common malignancy in Curcuma wenyujin. In the present study, we investigated the the world and the second leading cause of cancer-related anticancer effects of β-elemene in gastric cancer cells and the mortality (1). At present, surgical resection remains the potential proteins involved. Human SGC7901 and MKN45 main therapeutic strategy for gastric cancer, supplemented gastric cancer cells were treated with different concentrations with perioperative chemotherapy, chemoradiotherapy and/or of β-elemene. Cell viability, clonogenic survival and apoptotic immunotherapy (2-5). However, most patients are diagnosed cell death were assessed. β-elemene inhibited viability and with advanced gastric cancer which may have progressed decreased clonogenic survival of gastric cancer cells in a beyond the curative potential of surgical operation (6,7). In dose-dependent manner. Apoptosis induction contributed to addition, previous studies have demonstrated that a consid- the anticancer effects. We then employed a proteomic method, erable proportion of patients receiving potentially curative isobaric tags for relative and absolute quantitation (iTRAQ), to resection experienced recurrences which lead to unfavorable detect the proteins altered by β-elemene. In total, 147 upregu- prognosis (8,9). Adjuvant therapy, such as chemotherapy, lated proteins and 86 downregulated proteins were identified provides rather limited survival advantage (10,11). These facts in response to β-elemene treatment in SGC7901 gastric cancer attest to the deficiency in the current strategies for treating cells. Among them, expression of p21-activated gastric cancer and the demand for novel approaches to the kinase‑interacting protein 1 (PAK1IP1), Bcl-2-associated tran- management of gastric cancer. scription factor 1 (BTF) and topoisomerase 2-α (TOPIIα) were Among various ingredients eligible for adjuvant therapy validated by western blot analyses and the trends were consis- for gastric cancer, the significance of natural products, tent with iTRAQ results. Top pathways involved in β-elemene particularly the essence extracted from Chinese herbs are treatment in SGC7901 gastric cancer cells included ribo- gaining increasing attention in basic and clinical research (12). some signaling, peroxisome proliferator-activated receptors β-elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane) (PPARs) signaling pathway, regulation of actin cytoskeleton, is a novel anticancer agent extracted from the Chinese medicinal phagosome, biosynthesis and metabolism of some amino acids. herb Curcuma wenyujin (13). In recent studies, β-elemene was Collectively, our results suggest a promising therapeutic role shown to have diverse anticancer potential, such as inhibiting of β-elemene in gastric cancer. The differentially expressed proliferation and inducing apoptosis of cancer cells, and inter- proteins provide further insight into the potential mechanisms acting with multiple oncogenic or tumor suppressing signaling involved in gastric cancer treatment using β-elemene. pathways in a broad spectrum of cancers (14-16). Other studies found that β-elemene could enhance tumor chemosensitivity or overcome drug resistance (17,18). In addition, β-elemene has been approved by the China Food and Drug Administration as a therapeutic drug in clinical practice where its efficacy has been exhibited when combined with first‑line chemotherapy Correspondence to: Professor Xiang-Ming Che, Department of for malignant tumors (19,20). However, the mechanisms by General Surgery, The First Affiliated Hospital of Medical College of which β-elemene is involved in tumor suppressing activities Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China remain largely unknown. E-mail: [email protected] In the present study, we examined the anticancer potential of β-elemene in the proliferation, clonogenic survival and Key words: β-elemene, gastric cancer, proliferation, apoptosis, apoptosis in SGC7901 and MKN45 gastric cancer cells. Then, proteomics in order to investigate the molecules through which β-elemene exhibited its anticancer effects and to obtain a better under- standing of its therapeutic role in gastric cancer, we employed 2636 LIU et al: proteomic analysis of anticancer effects of β-elemene in gastric cancer cells isobaric tags for relative and absolute quantitation (iTRAQ), a number of cells seeding x PE). This assay was carried out in high-throughput proteomic approach, to profile proteins that duplicate. were differentially expressed following β-elemene treatment in gastric cancer cells. Protein preparation. After SGC7901 cells were treated with or without β-elemene at 30 µg/ml for 48 h, cell total protein Materials and methods was extracted. Protein concentration was determined using Pierce™ BCA Protein Assay (Thermo Scientific, Rockford, Reagents. β-elemene was obtained from Jingang Pharma­ IL, USA). Total protein extracted from two separate experi- ceutical Co. (Dalian, China). Annexin V-FITC/PI apoptosis ments was mixed together for use in the subsequent proteomic detection kit was purchased from 7 Sea Pharmacy Technology analysis. Protein samples were reduced and alkylated, then (Shanghai, China). iTRAQ reagents were from Applied added into 5-fold volume of ice-cold acetone and put in -20˚C Biosystems (New York, NY, USA). Anti-PAK1IP1 antibody condition overnight. Then, the precipitate was harvested by was purchased from Abcam (#ab67348; UK). Anti-TOPIIα centrifugation at 25,000 x g at 4˚C for 20 min and dried in the antibody was obtained from Proteintech (#20233-1-AP; USA). air for 5 min. The precipitate was dissolved in 200 µl 0.5 M Anti-BTF antibody was from BD Biosciences Pharmingen tetraethylammonium bromide (TEAB) and dealt with sonicate (#611726; San Diego, CA, USA). for 15 min. Finally, the supernatant was harvested and quanti- fied. Cell culture. The SGC7901 and MKN45 human gastric cancer cell lines were obtained from the Lab Animal Centre of the iTRAQ proteomic analysis. One hundred micrograms of protein Fourth Military Medical University (Xi'an, China). Cells were were taken out from each sample and digested with trypsin. cultured in RPMI-1640 medium (HyClone, USA), supple- Thereafter, the peptides of each sample were labeled with mented with 10% fetal bovine serum (Sijiqing, Huzhou City, iTRAQ reagents respectively, according to the manufacturer's

China) at 37˚C with 5% CO2 in a humidified atmosphere. protocol (Applied Biosystems) (SGC7901-control-114 tag and SGC7901-β-elemene treated-115 tag). Then, the labeled MTT assay. Cell viability was measured using 3-(4,5-dimethyl­ samples were pooled and sent to fractionating using strong thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. cation exchange (SCX) chromatography (Shimadzu LC-20AB The cells were seeded in 24-well plates at 5-10x104/well. After HPLC Pump system and the 4.6x250 mm Ultremex SCX overnight incubation, cells were exposed to different concen- column). After elution, 20 fractions of peptides were obtained. trations of β-elemene for 24-72 h. Then, 50 µl MTT (5 mg/ml) Each fraction was then desalted by Strata-X C18 column was added to each well and the cells were incubated for another (Phenomenex) and vacuum-dried. Each fraction of peptides 4 h at 37˚C. After gentle removal of the supernatant, 500 µl was resuspended in buffer A (5% ACN, 0.1% FA) and dimethyl sulfoxide (DMSO) was added to each well to solu- centrifuged at 20,000 x g for 10 min to remove the insoluble bilize the purple formazan crystal. The optical density (OD) substances. In each fraction, the final concentration of peptides was measured using a microplate reader at 490 nm and then was ~0.5 µg/µl. Five microlitres (~2.5 µg) of supernatant transformed into cell viability using the following formula: was loaded onto a Shimadzu LC-20AD nano HPLC by the Cell viability = (OD of the experimental sample)/(OD of the autosampler for separation. Mass spectrometric analysis of the control sample) x 100%. iTRAQ labelled peptides was performed using a Q Exactive (Thermo Fisher Scientific, San Jose, CA, USA) coupled online Annexin V-FITC/PI apoptosis detection assay. To explore the to the HPLC. Data processing of LC-MS/MS samples was effect of β-elemene on apoptotic cell death, Annexin V-FITC/PI searched against the International Protein Index (IPI) human apoptosis detection assay was used. The cells were seeded in protein database version 3.87 FASTA (91,464 sequences) using 6-well plates at 3x105/well. After overnight incubation, cells Mascot 2.3.02 software (Matrix Science, UK). When the fold- were exposed to different concentrations of β-elemene for change of protein abundance was >1.2 and the P-value was 24 h. Then, cells were collected and manipulated following the <0.05, we defined this protein as differentially expressed. The manufacturer's instructions, incubated with Annexin V-FITC identified proteins were categorized according to the and propidium iodide (PI), then analyzed using flow cytometry Ontology (GO) classification terms (http://www.geneontology. (FCM; BD Biosciences‑Clontech, Palo Alto, CA, USA) within org/). GO enrichment analysis was performed to display the 30 min. GO terms which the differentially expressed proteins enriched in all identified proteins. Clonogenic survival assay. Cells were trypsinized and counted. Then, 200 cells were seeded into each well of Western blot analyses. Equal amounts of protein samples 6-well plates. After overnight incubation for attachment and were subjected to SDS-PAGE. Proteins were transferred to the recovery, the cells were treated with different concentra- nitrocellulose (NC) membranes followed by 2 h blocking with tions of β-elemene. Ten to fourteen days after seeding, cells 5% skimmed milk at room temperature. The NC membranes were washed with PBS twice, fixed with methyl alcohol for were sequentially incubated with primary antibodies at 4˚C 15 min and stained with 1% crystal violet for 20 min. Colonies overnight and secondary antibody for 1 h at room temperature. containing >50 cells was counted and the surviving fractions The reactions were visualized using electrochemilumines- were calculated as follows: Plating efficiency (PE) = colony cence (ECL) detection kit (CWBIO, Beijing, China). The band number of the control group/the number of cells seeding. quantification was performed using Image-Pro Plus 6.0 soft- Surviving fraction = colony number of the treated-group/(the ware (Media Cybernetics). ONCOLOGY REPORTS 32: 2635-2647, 2014 2637

Figure 1. β-elemene inhibits the viability of gastric cancer cells. (A) SGC7901 and (B) MKN45 gastric cancer cells were treated with different concentrations of β-elemene (0, 10, 20, 30, 40, 60, 80 and 120 µg/ml) for 24, 48 or 72 h. Cell viability was determined using MTT assay. Dots and bars, means ± SD.

Figure 2. β-elemene induces apoptotic cell death in gastric cancer cells. Cells were treated with different concentrations of β-elemene for 24 h. Apoptotic rate was analyzed by FCM following the manufacturer's instructions. (A) Representative FCM results of SGC7901 cells. a, control; b, 20 µg/ml; c, 30 µg/ml; d, 40 µg/ml; e, 60 µg/ml; and f, 80 µg/ml. (B) Student's t-test of apoptotic rate was performed between the control group and groups treated with β-elemene in SGC7901 cells. (C) Representative FCM results of MKN45 cells. a, control; b, 10 µg/ml; c, 20 µg/ml; d, 30 µg/ml; e, 40 µg/ml; and f, 60 µg/ml. (D) Quantification of apoptosis of the control group and groups treated with β-elemene in MKN45 cells. *P<0.05. Column and bars, means ± SD. FCM, flow cytometry.

Statistical analysis. Data are presented as the means ± SD. Results Statistical analysis was performed using a two-tailed Student's t-test through SPSS 17.0 software (Chicago, USA). P-value β-elemene inhibits the viability of gastric cancer cells. To <0.05 was considered to indicate a statistically significant investigate the antiproliferative effect of β-elemene in gastric difference. cancer cells, SGC7901 and MKN45 cells were exposed to 2638 LIU et al: proteomic analysis of anticancer effects of β-elemene in gastric cancer cells

gastric cancer cells at 24, 48 and 72 h were 67.15, 56.89 and

46.05 µg/ml, respectively. The IC50 values for MKN45 cells at 24, 48 and 72 h were 45.57, 37.97 and 35.29 µg/ml, respectively. These results indicate that β-elemene inhibits the viability of gastric cancer cells in a dose-dependent manner.

β-elemene induces apoptotic cell death in gastric cancer cells. FCM analysis showed that the apoptotic rate gradually increased after 24-h exposure to increased concentrations of β-elemene. The percentage of apoptotic cells in SGC7901 cells in the control group and β-elemene-treated groups (20, 30, 40, 60 and 80 µg/ml) was 8.5±0.5, 10.4±1.0, 22.2±1.6, 32.6±5.4, 42.8±4.3 and 52.1±4.3%, respectively (Fig. 2A and B). Compared with the control group, the increased rate of apoptosis reached statis- tical significance when the concentrations of β-elemene were >30 µg/ml in SGC7901 cells. Similar trends of apoptosis induc- tion effects were observed in MKN45 cells (Fig. 2C and D). These data suggest that β-elemene induces apoptotic cell death in gastric cancer cells in a dose-dependent manner.

β-elemene decreases clonogenic survival of gastric cancer cells. To determine whether β-elemene inhibits colony forming efficiency, cells were exposed to different concen- trations of β-elemene (0, 10, 20, 30, 40 and 60 µg/ml) and grown in a cell contact-independent manner. Consistent with the observed effects on cell viability and apoptosis-induction activity, β-elemene exhibited anti-clonogenic potential and led to a statistically significant reduction in colony formation (Fig. 3). It is worth noting that the size of the colonies tended to be smaller after treatment with β-elemene (Fig. 3B). When the concentration of β-elemene reached 60 µg/ml, the number of survived cells in each cloned cell group barely reached the standard of a colony in SGC7901 cells. These results suggest that β-elemene induces a dose-dependent inhibition of clono- genicity in gastric cancer cells.

iTRAQ identification and quantification of differentially expressed proteins by β-elemene in SGC7901 gastric cancer cells. Through iTRAQ analysis, 17,154 unique peptides corre- sponding to 4,267 proteins were identified in SGC7901 gastric cancer cells (data not shown). According to our definition of differentially expressed protein, a total of 233 identified proteins were regulated by β-elemene intervention in SGC7901 gastric cancer cells, including 147 upregulated proteins and 86 downregulated proteins. The altered proteins of both lists are Figure 3. β-elemene inhibits clonogenic survival of SGC7901 gastric cancer cells. Two hundred single cells were seeded to each well of the 6-well plates. shown in Tables I and II, respectively. After attachment and recovery, cells were treated with or without β-elemene and incubated for 10-14 days to form typical colonies. (A) The entire appear- GO analysis of differentially expressed proteins altered by ance of representative wells of each experimental group. (B) Appearance of β-elemene in SGC7901 gastric cancer cells. Based on the GO representative colony from each experimental group. (C) Clonogenic survival of the control group and groups treated with β-elemene. The results are terms analysis, categorization of these differentially expressed representative of duplicate independent experiments. a, control; b, 10 µg/ml; proteins according to cellular component, molecular function c, 20 µg/ml; d, 30 µg/ml; e, 40 µg/ml; and f, 60 µg/ml. *P<0.05. Column and and biological process is shown in Fig. 4. GO enrichment bars, means ± SD. analysis shows the top pathways involved in the differentially expressed proteins resulting from β-elemene treatment. They include phenylalanine, tyrosine and tryptophan biosynthesis, different concentrations of β-elemene (0, 10, 20, 30, 40, 60, 80 signaling, tyrosine metabolism, phenylalanine and 120 µg/ml) for 24, 48 or 72 h. Cell viability analysis showed metabolism, PPAR signaling pathway, regulation of actin that β-elemene suppressed the viability of gastric cancer cytoskeleton, cysteine and methionine metabolism, ether cells in a dose-dependent manner (Fig. 1). The 50% inhibi- lipid metabolism, hematopoietic cell lineage and phagosome tory concentration (IC50) values of β-elemene for SGC7901 signaling pathways. ONCOLOGY REPORTS 32: 2635-2647, 2014 2639

Table I. Upregulated proteins in response to β-elemene treatment in SGC7901 gastric cancer cells.

Gene Fold-ratio No. Accession symbol Protein name (115:114)

1 IPI00549540 PAK1IP1 p21-activated protein kinase-interacting protein 1 3.967 2 IPI00152429 SPNS1 Isoform 3 of protein spinster homolog 1 2.979 3 IPI00021885 FGA Isoform 1 of fibrinogen α chain 2.397 4 IPI00011200 PHGDH D-3-phosphoglycerate dehydrogenase 2.319 5 IPI00006213 PCM1 Isoform 1 of pericentriolar material 1 protein 2.316 6 IPI00418426 CNNM4 Metal transporter CNNM4 2.313 7 IPI00013809 GSTZ1 Isoform 1 of maleylacetoacetate isomerase 2.292 8 IPI00012433 F8A Factor VIII intron 22 protein 2.172 9 IPI00015856 DNPEP Aspartyl aminopeptidase 2.144 10 IPI00002564 XRCC1 DNA repair protein XRCC1 1.981 11 IPI00909584 HARS2 Histidyl-tRNA synthetase homolog 1.922 12 IPI00218116 OASL Isoform p30 of 59 kDa 2'-5'-oligoadenylate synthase-like protein 1.915 13 IPI00216138 TAGLN Transgelin 1.904 14 IPI00829741 ANKLE2 Isoform 1 of ankyrin repeat and LEM domain-containing protein 2 1.872 15 IPI00022145 NUCKS1 Isoform 1 of nuclear ubiquitous casein and cyclin-dependent 1.771 kinases substrate 16 IPI00290979 ABHD5 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 1.761 17 IPI00554777 ASNS Asparagine synthetase (glutamine-hydrolyzing) 1.664 18 IPI00021978 PEX11B Peroxisomal membrane protein 11B 1.661 19 IPI00328737 ZNF598 Isoform 1 of Zinc finger protein 598 1.644 20 IPI00101782 GMPPA Isoform 1 of mannose-1-phosphate guanyltransferase α 1.641 21 IPI00007320 TCF25 Transcription factor 25 1.627 22 IPI00031651 C7orf50 Uncharacterized protein C7orf50 1.623 23 IPI00247583 RPL21 60S L21 1.622 24 IPI00329596 TMX2 Uncharacterized protein 1.613 25 IPI00740961 INTS1 DKFZP586J0619 protein 1.568 26 IPI00102752 RBM15 Isoform 1 of putative RNA-binding protein 15 1.544 27 IPI00024650 SLC16A1 Monocarboxylate transporter 1 1.539 28 IPI00026848 LRPAP1 α-2-macroglobulin receptor-associated protein 1.534 29 IPI00019976 CCDC85B Coiled-coil domain-containing protein 85B 1.52 30 IPI00021831 PRKAR1A cAMP-dependent protein kinase type I-α regulatory subunit 1.485 31 IPI00145260 IBA57 Putative transferase CAF17, mitochondrial 1.474 32 IPI00550021 RPL3 1.467 33 IPI00853369 PLXNB2 Plexin-B2 1.465 34 IPI00001734 PSAT1 Phosphoserine aminotransferase 1.463 35 IPI00412607 RPL35 60S ribosomal protein L35 1.449 36 IPI00027463 S100A6 Protein S100-A6 1.447 37 IPI00012772 RPL8 1.431 38 IPI00299219 CYR61 Protein CYR61 1.425 39 IPI00006362 EDF1 Isoform 2 of endothelial differentiation-related factor 1 1.423 40 IPI00007309 TIMM23 Mitochondrial import inner membrane translocase subunit Tim23 1.422 41 IPI00030362 PLP2 Isoform 1 of proteolipid protein 2 1.421 42 IPI00219840 AP2S1 Isoform 1 of AP-2 complex subunit sigma 1.42 43 IPI00980827 Unknown Uncharacterized protein 1.419 44 IPI00296526 NAGK N-acetyl-D-glucosamine kinase 1.417 45 IPI00465361 RPL13 60S ribosomal protein L13 1.416 46 IPI00306749 SLC4A1AP Kanadaptin 1.409 47 IPI00168262 GLT25D1 Procollagen galactosyltransferase 1 1.404 48 IPI00410067 ZC3HAV1 Isoform 1 of Zinc finger CCCH-type antiviral protein 1 1.397 49 IPI00789805 DIAPH3 Isoform 3 of protein diaphanous homolog 3 1.395 50 IPI00293425 FXN Isoform 1 of frataxin, mitochondrial 1.394 2640 LIU et al: proteomic analysis of anticancer effects of β-elemene in gastric cancer cells

Table I. Continued.

Gene Fold-ratio No. Accession symbol Protein name (115:114)

51 IPI00021389 CCS Copper chaperone for superoxide dismutase 1.376 52 IPI00027096 MRPL19 39S ribosomal protein L19, mitochondrial 1.376 53 IPI00005040 ACADM Isoform 1 of medium-chain specific acyl-CoA 1.376 dehydrogenase, mitochondrial 54 IPI00556655 LAMP1 LAMP1 protein variant (fragment) 1.375 55 IPI00101968 DBNL Isoform 3 of drebrin-like protein 1.37 56 IPI00008418 DIABLO Diablo homolog, mitochondrial precursor 1.36 57 IPI00396174 CCDC25 Coiled-coil domain-containing protein 25 1.357 58 IPI00010404 SF3B5 Splicing factor 3B subunit 5 1.356 59 IPI00216999 C14orf21 Pumilio domain-containing protein C14orf21 1.354 60 IPI00293276 MIF Macrophage migration inhibitory factor 1.352 61 IPI00297241 URB1 Nucleolar pre-ribosomal-associated protein 1 1.351 62 IPI00004406 UPP1 Isoform 1 of uridine phosphorylase 1 1.347 63 IPI00023122 PDLIM7 Isoform 1 of PDZ and LIM domain protein 7 1.346 64 IPI00375731 RBM10 Isoform 1 of RNA-binding protein 10 1.345 65 IPI00170786 WBP11 WW domain-binding protein 11 1.345 66 IPI00010740 SFPQ Isoform long of splicing factor, proline- and glutamine-rich 1.343 67 IPI00329321 LYRM7 LYR motif-containing protein 7 1.341 68 IPI00290857 KRT3 Keratin, type II cytoskeletal 3 1.34 69 IPI00456758 RPL27A 60S ribosomal protein L27a 1.339 70 IPI00063673 ISY1 Isoform 1 of pre-mRNA-splicing factor ISY1 homolog 1.339 71 IPI00217236 TBCA Tubulin-specific chaperone A 1.339 72 IPI00301280 TMEM43 Transmembrane protein 43 1.337 73 IPI00006079 BCLAF1 Isoform 1 of Bcl-2-associated transcription factor 1 1.336 74 IPI00746351 DIS3 Isoform 1 of exosome complex exonuclease RRP44 1.33 75 IPI00180781 MLKL Isoform 1 of mixed lineage kinase domain-like protein 1.328 76 IPI00012493 RPS20 40S ribosomal protein S20 1.321 77 IPI00008922 IFITM2 Interferon-induced transmembrane protein 2 1.32 78 IPI00647650 EIF3H Eukaryotic 3 subunit 3 1.317 79 IPI00299573 RPL7A 60S ribosomal protein L7a 1.317 80 IPI01012991 LUC7L2 Isoform 1 of putative RNA-binding protein Luc7-like 2 1.315 81 IPI00300078 PWP2 Periodic tryptophan protein 2 homolog 1.311 82 IPI00003016 STRN4 Striatin-4 1.31 83 IPI00745955 EBNA1BP2 Probable rRNA-processing protein EBP2 1.307 84 IPI00220527 SNX1 Isoform 1A of sorting nexin-1 1.305 85 IPI00017448 RPS21 40S ribosomal protein S21 1.3 86 IPI00002135 TACC3 Transforming acidic coiled-coil-containing protein 3 1.296 87 IPI00011635 BCL2L13 Isoform 2 of Bcl-2-like protein 13 1.291 88 IPI00157176 MEA1 Male-enhanced antigen 1 1.288 89 IPI00012756 IFIT5 Interferon-induced protein with tetratricopeptide repeats 5 1.287 90 IPI00022202 SLC25A3 Isoform A of phosphate carrier protein, mitochondrial 1.287 91 IPI00064767 ARHGAP17 Isoform 1 of Rho GTPase-activating protein 17 1.284 92 IPI00063827 ABHD14B Isoform 1 of abhydrolase domain-containing protein 14B 1.282 93 IPI00243221 NRD1 Nardilysin isoform a 1.274 94 IPI00297160 CD44 Isoform 12 of CD44 antigen 1.272 95 IPI00014808 PAFAH1B3 Platelet-activating factor acetylhydrolase IB subunit γ 1.269 96 IPI00022694 PSMD4 Isoform Rpn10A of 26S proteasome non-ATPase regulatory subunit 4 1.264 97 IPI00011229 CTSD Cathepsin D 1.261 98 IPI00003856 ATP6V1E1 V-type proton ATPase subunit E 1 1.255 99 IPI00291669 UBLCP1 Ubiquitin-like domain-containing CTD phosphatase 1 1.252 ONCOLOGY REPORTS 32: 2635-2647, 2014 2641

Table I. Continued.

Gene Fold-ratio No. Accession symbol Protein name (115:114)

100 IPI00029081 LIG3 Isoform α of DNA ligase 3 1.248 101 IPI00007797 FABP5 Fatty acid-binding protein, epidermal 1.248 102 IPI00028387 DDRGK1 Isoform 1 of DDRGK domain-containing protein 1 1.245 103 IPI00007694 PPME1 Isoform 1 of protein phosphatase methylesterase 1 1.243 104 IPI00031519 DNMT1 Isoform 1 of DNA (cytosine-5)-methyltransferase 1 1.241 105 IPI00219684 FABP3 Fatty acid-binding protein, heart 1.24 106 IPI00219153 RPL22 60S ribosomal protein L22 1.237 107 IPI00219029 GOT1 Aspartate aminotransferase, cytoplasmic 1.236 108 IPI00297178 DHX16 119 kDa protein 1.234 109 IPI00305267 GOLGA3 Isoform 1 of golgin subfamily A member 3 1.234 110 IPI00010697 ITGA6 Isoform α-6X1X2B of integrin α-6 1.233 111 IPI00396387 GNL1 Isoform 1 of guanine nucleotide-binding protein-like 1 1.233 112 IPI00000030 PPP2R5D Isoform δ-1 of serine/threonine-.233 2A 56 kDa regulatory subunit δ isoform 113 IPI00007118 SERPINE1 Plasminogen activator inhibitor 1 1.23 114 IPI00021057 SLC12A4 Isoform 1 of solute carrier family 12 member 4 1.229 115 IPI00018206 GOT2 Aspartate aminotransferase, mitochondrial 1.229 116 IPI00019472 SLC1A5 Neutral amino acid transporter B(0) 1.229 117 IPI00306353 DUSP23 Dual specificity protein phosphatase 23 1.227 118 IPI00032825 TMED7 Transmembrane emp24 domain-containing protein 7 1.227 119 IPI01010270 WIZ WIZ protein 1.225 120 IPI00006980 C14orf166 UPF0568 protein C14orf166 1.224 121 IPI00298625 LYN Isoform LYN A of tyrosine-protein kinase Lyn 1.223 122 IPI00550523 ATL3 Atlastin-3 1.222 123 IPI00333215 TCEA1 Isoform 1 of transcription A protein 1 1.222 124 IPI00032038 CPT1A Isoform 1 of carnitine O-palmitoyltransferase 1, liver isoform 1.221 125 IPI00025512 HSPB1 Heat shock protein β-1 1.221 126 IPI00013468 BUB3 Isoform 1 of mitotic checkpoint protein BUB3 1.221 127 IPI00307165 TRIM47 Tripartite motif-containing protein 47 1.22 128 IPI00181396 VPRBP Isoform 3 of protein VPRBP 1.217 129 IPI00216237 RPL36 60S ribosomal protein L36 1.217 130 IPI00550069 RNH1 Ribonuclease inhibitor 1.217 131 IPI00215995 ITGA3 Isoform 1 of integrin α-3 1.216 132 IPI00902512 PPP1CA Serine/threonine-protein phosphatase 1.215 133 IPI00003923 UMPS Isoform 1 of uridine 5'-monophosphate synthase 1.21 134 IPI00010414 PDLIM1 PDZ and LIM domain protein 1 1.21 135 IPI01026021 SHMT2 SHMT2 protein 1.21 136 IPI00021828 CSTB Cystatin-B 1.21 137 IPI00060627 CCDC124 Coiled-coil domain-containing protein 124 1.209 138 IPI00218466 SEC61A1 Protein transport protein Sec61 subunit α isoform 1 1.207 139 IPI00026546 PAFAH1B2 Platelet-activating factor acetylhydrolase IB subunit β 1.207 140 IPI00011592 DYNC1LI2 Cytoplasmic dynein 1 light intermediate chain 2 1.206 141 IPI00216682 CNN3 Calponin-3 1.206 142 IPI00297900 DDX10 Probable ATP-dependent RNA helicase DDX10 1.206 143 IPI00292657 PTGR1 Prostaglandin reductase 1 1.204 144 IPI00925046 QARS Glutaminyl-tRNA synthetase 1.204 145 IPI00339379 ARHGEF1 Isoform 2 of Rho guanine nucleotide exchange factor 1 1.202 146 IPI00294486 DUSP9 Dual specificity protein phosphatase 9 1.201 147 IPI00159899 ANKFY1 Isoform 1 of ankyrin repeat and FYVE domain-containing protein 1 1.201 2642 LIU et al: proteomic analysis of anticancer effects of β-elemene in gastric cancer cells

Table II. Downregulated proteins in response to β-elemene treatment in SGC7901 gastric cancer cells.

Gene Fold-ratio No. Accession symbol Protein name (115:114)

1 IPI00654755 HBB Hemoglobin subunit β 0.163 2 IPI00290380 ALPPL2 Alkaline phosphatase, placental-like 0.531 3 IPI00183695 S100A10 Protein S100-A10 0.598 4 IPI00419273 CUL4A Isoform 1 of cullin-4A 0.631 5 IPI00410110 DHX40 Isoform 1 of probable ATP-dependent RNA helicase DHX40 0.637 6 IPI00011698 SAP18 Histone deacetylase complex subunit SAP18 0.654 7 IPI00022002 MRPS27 Mitochondrial 28S ribosomal protein S27 0.664 8 IPI00171438 TXNDC5 Thioredoxin domain-containing protein 5 0.675 9 IPI00023704 LPP Lipoma-preferred partner 0.701 10 IPI00301503 TRA2B Isoform 1 of transformer-2 protein homolog β 0.706 11 IPI00414836 OSTF1 Osteoclast-stimulating factor 1 0.706 12 IPI00019896 MYCBP2 Homo sapiens protein associated with Myc mRNA (fragment) 0.712 13 IPI00414101 TOP2A Isoform 2 of DNA topoisomerase 2-α 0.716 14 IPI00412545 NDUFA5 NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 5 0.717 15 IPI00003935 HIST2H2BE Histone H2B type 2-E 0.718 16 IPI00291467 SLC25A6 ADP/ATP translocase 3 0.722 17 IPI00374272 C5orf51 UPF0600 protein C5orf51 0.725 18 IPI00005087 TMOD3 Tropomodulin-3 0.732 19 IPI00008475 HMGCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic 0.736 20 IPI00017342 RHOG Rho-related GTP-binding protein RhoG 0.742 21 IPI00009901 NUTF2 Nuclear transport factor 2 0.745 22 IPI00298731 PPP1R10 Serine/threonine-protein phosphatase 1 regulatory subunit 10 0.746 23 IPI00020602 CSNK2A2 Casein kinase II subunit α 0.753 24 IPI00025347 EMG1 Ribosomal RNA small subunit methyltransferase NEP1 0.755 25 IPI00218962 C20orf43 UPF0549 protein C20orf43 0.755 26 IPI00014230 C1QBP Complement component 1 Q subcomponent-binding protein, 0.755 mitochondrial 27 IPI00219025 GLRX Glutaredoxin-1 0.757 28 IPI00168479 APOA1BP Apolipoprotein A-I binding protein 0.758 29 IPI00027705 PRIM2 Isoform 1 of DNA primase large subunit 0.758 30 IPI00219483 SNRNP70 Isoform 2 of U1 small nuclear ribonucleoprotein 70 kDa 0.765 31 IPI00220014 IDI1 Isoform 2 of isopentenyl-diphosphate δ-isomerase 1 0.766 32 IPI00434580 MYOM1 Isoform 1 of myomesin-1 0.771 33 IPI00023729 FN3K Fructosamine-3-kinase 0.773 34 IPI00013446 PSCA Prostate stem cell antigen 0.78 35 IPI00924816 MTPN Myotrophin 0.781 36 IPI00033022 DNM2 Isoform 1 of dynamin-2 0.781 37 IPI00009032 SSB Lupus la protein 0.781 38 IPI00073779 MRPS35 Isoform 1 of 28S ribosomal protein S35, mitochondrial 0.782 39 IPI00163644 OSBPL8 Oxysterol-binding protein 0.783 40 IPI00419626 MRPL55 Isoform 2 of 39S ribosomal protein L55, mitochondrial 0.789 41 IPI00006440 MRPS7 28S ribosomal protein S7, mitochondrial 0.79 42 IPI00027704 PRIM1 DNA primase small subunit 0.792 43 IPI00018768 TSN Translin 0.795 44 IPI00876962 INF2 Isoform 2 of inverted formin-2 0.795 45 IPI00156374 IPO4 Isoform 1 of importin-4 0.795 46 IPI00017344 RAB5B Ras-related protein Rab-5B 0.799 47 IPI00418290 MRPL14 39S ribosomal protein L14, mitochondrial 0.8 48 IPI00022820 GTF2B Transcription initiation factor IIB 0.801 49 IPI00006579 COX4I1 Cytochrome c oxidase subunit 4 isoform 1, mitochondrial 0.801 50 IPI00217766 SCARB2 Lysosome membrane protein 2 0.801 ONCOLOGY REPORTS 32: 2635-2647, 2014 2643

Table II. Continued.

Gene Fold-ratio No. Accession symbol Protein name (115:114)

51 IPI00013396 SNRPC U1 small nuclear ribonucleoprotein C 0.802 52 IPI00022977 CKB Creatine kinase B-type 0.804 53 IPI00012074 HNRNPR Isoform 1 of heterogeneous nuclear ribonucleoprotein R 0.808 54 IPI00216171 ENO2 γ-enolase 0.809 55 IPI00014958 PON2 Isoform 1 of serum paraoxonase/arylesterase 2 0.812 56 IPI00018288 POLR2C DNA-directed RNA polymerase II subunit RPB3 0.812 57 IPI00010948 TRIM26 Tripartite motif-containing protein 26 0.812 58 IPI00007052 FIS1 Mitochondrial fission 1 protein 0.813 59 IPI00329625 TBRG4 Transforming growth factor β regulator 4 0.813 60 IPI00017510 MT-CO2 Cytochrome c oxidase subunit 2 0.814 61 IPI00645898 XPNPEP1 X-prolyl aminopeptidase (aminopeptidase P) 1, soluble 0.814 62 IPI00029054 NT5C2 Cytosolic purine 5'-nucleotidase 0.816 63 IPI00374970 SEPT10 Isoform 1 of septin-10 0.817 64 IPI00005948 MRI1 Isoform 1 of methylthioribose-1-phosphate isomerase 0.817 65 IPI00017526 S100P Protein S100-P 0.817 66 IPI00026964 UQCRFS1 Cytochrome b-c1 complex subunit Rieske, mitochondrial 0.817 67 IPI00219673 GSTK1 Isoform 1 of glutathione S-transferase κ 1 0.818 68 IPI00296432 IWS1 Isoform 1 of protein IWS1 homolog 0.818 69 IPI00029697 EXOSC9 Isoform 2 of exosome complex component RRP45 0.82 70 IPI00062336 RPRD1A Isoform 2 of regulation of nuclear pre-mRNA 0.822 domain-containing protein 1A 71 IPI00179172 PPFIBP1 Isoform 2 of liprin-β-1 0.822 72 IPI00015972 COX6C Cytochrome c oxidase subunit 6C 0.823 73 IPI00410657 RNMT Isoform 2 of mRNA cap guanine-N7 methyltransferase 0.823 74 IPI00029019 UBAP2L Isoform 2 of ubiquitin-associated protein 2-like 0.825 75 IPI00007188 SLC25A5 ADP/ATP translocase 2 0.826 76 IPI00292056 PIK3C2B Phosphatidylinositol-4-phosphate 3-kinase C2 0.828 domain-containing subunit β 77 IPI00013475 TUBB2A Tubulin β-2A chain 0.829 78 IPI00293590 MGLL Monoglyceride lipase isoform 1 0.829 79 IPI00303568 PTGES2 Prostaglandin E synthase 2 0.829 80 IPI00027444 SERPINB1 Leukocyte elastase inhibitor 0.829 81 IPI00003765 CAPN7 Calpain-7 0.83 82 IPI00215920 ARF6 ADP-ribosylation factor 6 0.831 83 IPI00008449 FIP1L1 Isoform 3 of pre-mRNA 3'-end-processing factor FIP1 0.831 84 IPI00061178 RBMXL1 Heterogeneous nuclear ribonucleoprotein G-like 1 0.831 85 IPI00180292 BAIAP2 Isoform 5 of brain-specific angiogenesis 0.832 inhibitor 1-associated protein 2 86 IPI00029468 ACTR1A α-centractin 0.833

Validation of iTRAQ results by western blot analyses. Discussion Western blot analyses were performed to validate the differ- entially expressed proteins discovered by iTRAQ proteomic Previous studies have shown that β-elemene, a promising anti- analysis. The SGC7901 human gastric cancer cells were cancer drug extracted from natural plants, has efficient growth treated the same as for the iTRAQ analysis and lysed for inhibition effects in a broad range of cancer cells, although protein samples. Three proteins were selected for valida- with slight toxicity to normal tissue cells (14,21,22). In China, it tion purposes according to our interests and the availability has been used as a therapeutic candidate for certain malignant of antibodies. The results were consistent with those found tumors for several years (20). However, little is known about using iTRAQ (Fig. 5) and indicated the high reliability of our the underlying molecules. In the present study, our data indi- iTRAQ results. cated that β-elemene efficiently suppressed the proliferation 2644 LIU et al: proteomic analysis of anticancer effects of β-elemene in gastric cancer cells

Figure 4. Categorization of the differentially expressed proteins after β-elemene treatment in SGC7901 human gastric cancer cells according to the GO terms system. (A) Protein classification according to the cellular component. (B) Protein classification according to molecular function. (C) Protein classification according to biological process. GO, .

and survival of gastric cancer cells at least partly through the β-elemene treatment in gastric cancer cells provided insight induction of apoptosis. The effects are consistent with results and supported the results found at the cytological level. in other malignancies (15,21). Different from other studies, we Furthermore, the analysis provided some other molecules and employed an iTRAQ proteomic method to explore the poten- signal pathways that may predict other pharmacologic actions tial proteins that may contribute to its anticancer effect. As of β-elemene that had not been studied in cancer therapy. In a result, the differentially expressed proteins in response to brief, our results provide evidence that β-elemene may be a ONCOLOGY REPORTS 32: 2635-2647, 2014 2645

hyperactivated in several types of cancer and play a critical role in tumorigenesis and metastasis. Inhibition of PAK1 may efficiently block the transformation of cancer cells and act as a therapeutic strategy in cancer treatment (30,32,33). As a negative regulator of PAK1, PAK1IP1 specifically binds to the N-terminal regulatory domain of PAK1 and inhibits the activation of PAK1 by blocking the binding site of Rac and Cdc42, thus playing a negative role in cancer development and progression (30,31). However, little research has focused on PAK1IP1. In a recent study, Yu et al found that PAK1IP1 was upregulated when cancer cells were suffering from ribo- somal stresses and overexpression of PAK1IP1 could inhibit Figure 5. Validation of iTRAQ proteomic results by western blot analyses proliferation via p53-MDM2 loop (34). In the present study, in SGC7901 gastric cancer cells. After exposure to β-elemene at 30 µg/ml PAK1IP1 expression was ~3-fold upregulated in β-elemene- for 48 h, the expression levels of PAK1IP1, BTF and TOPIIα were analyzed treated gastric cancer cells. Therefore, we hypothesize that by western blot analyses. The trends closely match the iTRAQ proteomic β-elemene may upregulate PAK1IP1 expression and thus results. iTRAQ, isobaric tags for relative and absolute quantitation; PAK1IP1, p21-activated protein kinase‑interacting protein 1; BTF, Bcl-2-associated inhibit the activation of PAK1, which subsequently inhibits transcription factor 1; TOPIIα, topoisomerase 2-α. proliferation and induces apoptosis in gastric cancer cells. One of the major protein groups regulated by β-elemene in SGC7901 gastric cancer cells was ribosomal proteins, with 12 ribosomal proteins upregulated and 4 ribosomal proteins potential drug for gastric cancer. Some of the key proteins are downregulated. Over the last decade, some ribosomal proteins potential markers of gastric cancer treatment and are briefly have been linked with emerging functions in cancer, in addi- discussed below. tion to protein synthesis. Ribosomal protein L5 (RPL5), Bcl-2 family proteins were found to be modulated in along with RPL11 and RPL23, may form a complex with β-elemene-treated cancer cells in previous studies and make MDM2 oncoprotein and activate p53 through the inhibition sense in apoptosis induction (14,21,22). The balance between of MDM2-mediated p53 degradation (35). RPS7 was also pro-apoptotic and anti-apoptotic members of the Bcl-2 family found to interact with MDM2 and overexpression of RPS7 proteins decides the fates of cells, and the increased propor- increased cell apoptosis and suppressed cell proliferation after tion of pro-apoptotic proteins results in apoptotic cell death. p53 activation (36). RPS14 and RPL11 were demonstrated Therefore, they play vital role in cancer therapy (23). In the to inhibit cell proliferation by negative regulation of c-Myc present study, Bcl-2-associated transcription factor 1 (BTF) activity (37,38). In some other studies, the correlation between and Bcl-2-like protein 13 (also known as Bcl-rambo) were ribosomal proteins and drug resistance in cancer therapy was upregulated to 1.34- and 1.29-fold in response to β-elemene established. RPS13 and RPL23 could suppress drug-induced treatment compared with the control untreated gastric cancer apoptosis and thus mediate multidrug resistance in gastric cells, respectively. Both play death-promoting roles in cancer cancer cells (39). RPL35a was found overexpressed in many cells. BTF was first identified as a transcriptional repressor glioblastoma multiforme (GBM) brain tumors and led to that interacted with Bcl-2 family proteins and overexpres- chemotherapy resistance in GBM (40). Together with the sion of BTF could induce apoptosis (24). Previous studies present study, these results propose more roles of ribosomal demonstrated the roles of BTF in apoptosis promotion through proteins in cancer and therefore merit further attention in the control of transcription or correlations with Bcl-2 family cancer therapy research. members (25-27). Moreover, BTF has been shown to inhibit S100A10 was a top molecule significantly downregulated DNA damage repair (25,27). This may partly contribute to the by β-elemene in the present study. S100A10, also known as p11, fact that β-elemene enhances tumor chemosensitivity or over- is a unique member of the S100 protein family which serves comes drug resistance (17,18). The other molecule, Bcl-rambo, for intracellular calcium signaling and is characterized by two is also a pro-apoptotic member of the Bcl-2 family (28,29). EF hand motifs (41,42). The homodimer of S100A10 forms a Although it was found to trigger cell death in a way distinct heterotetrameric complex with two molecules of Annexin A2, from the traditional Bcl-2 family members, Bcl-rambo- a type of plasma membrane protein, to maintain stability and induced apoptotic signaling pathway eventually joined other execute its functions (43). Expression of S100A10 has been pro-apoptotic pathways at the level of caspase-3 (28). Taken detected in a broad spectrum of tissues and cancers including together, these results indicate that Bcl-2 family proteins play a gastric cancer (44-47). Over the past years, increasing evidence critical role in β-elemene-induced cell death in gastric cancer has demonstrated the promoting role of S100A10 in tumor cells. invasion and metastasis and that knockdown of S100A10 p21-activated protein kinase-interacting protein 1 (PAK1IP1) could efficiently suppress cancer progression (46-48). Notably, was the most influenced protein among the list of upregulated PAK1IP1, the most influenced protein of upregulated proteins proteins by β-elemene. P21-activated protein kinase 1 (PAK1) by β-elemene, specifically targets PAK1 which is also most and PAK signal pathways have been shown to have multiple associated with cytoskeletal dynamics. Collectively, we roles in cancer cell biological behaviors, such as cytoskeletal deduced that β-elemene may inhibit invasion and metastasis dynamics, survival, proliferation and transcription (30,31). in gastric cancer therapy, which warrants further investiga- PAK1 and its PAK family members are overexpressed or tion. 2646 LIU et al: proteomic analysis of anticancer effects of β-elemene in gastric cancer cells

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